Method and agent for the enzymatic determination of glucose



United States Patent ()1 fice METHOD AND AGENT FOR THE ENZYMATIC DETERMINATION OF GLUCOSE Hans Ulrich Bergmeyer and Hans Moellering, Tutzing,

Upper Bavaria, Germany, assignors to Boehringer Mannheim Gesellschaft mit beschrankter Haftung, a corporation of Germany No Drawing. Filed Aug. 10, 1966, Ser. No. 571,416 Claims priority, application Germany, Aug. 17, 1965,

Int. Cl. G01n 31/14 US. Cl. 195-103.5 9 Claims ABSTRACT OF THE DISCLOSURE Process for the enzymatic determination of glucose comprising mixing a sample containing glucose with a phosphate donor having the formula:

This invention relates to a method and agent for use in the enzymatic determination of glucose.

Analytical methods involving enzymes are, in general, characterized by their high substrate specificity and allow for the exact determination of the substrate in the presence of most accompanying materials which are characterized by similar constitution and which represent disturbing fac tors in the same determination using non-enzymatic analytical methods.

Enzymatic methods for the determination of glucose have also already been proposed and introduced into practice and include, for example, the reaction of glucose with glucose oxidase yielding D-gluconic acid and hydrogen peroxide and the reaction with hexokinase and adenosine triphosphate producing glucose-6-phosphate. The reaction products produced, i.e., hydrogen peroxide and glucose-6- phosphate, thereupon are converted in a further reaction into products which can be measured colorimetrically or photometrically (of M. W. Slein in H. U. Bergmeyers Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim, 1962, page 117; and H. U. Bergmeyer and E. Bernt, loc. cit., page 123).

3,509,025 Patented Apr. 28, 1970 hexokinase glucose ATP G6P ADP G-G-P DH (In the above equations, ATP=adenosine-5'-triphosphoric acid; ADP=adenosine-5-diphosphoric acid; G-6- P=glucose-6-phosphoric acid; TPN and TPNH=triphosphopyridine nucleotide and its reduced form; 6-PG=6- phosphogluconic acid; and G-6-PDH=glucose-6-phosphate dehydrogenase.)

In spite of the excellent sensitivity and accuracy of the above-mentioned method, it has the disadvantage that extremely pure and, as a result, expensive enzyme preparations must be used as hexokinase also converts fructose into fructose-6-phosphate and mannose into mannose-6- phosphate. If the enzyme preparations used contain, for example, only traces of the enzyme phosphohexose isomerase, then any fructose present is converted into glucose-6-phosphate. This results in a false analytical determination of the glucose present.

In accordance with the invention, it has now, surprisingly, been found that the enzyme from Aerobacter aerogenes which was recently described by M. Y. Kamel and R. L. Anderson (J. Biol. Chem., 239, PC 3607, 1964) is outstandingly suitable for use in the enzymatic determination of glucose. In contradistinction to hexokinase, this enzyme substantially does not react with fructose. The relative reaction velocity of this new enzyme with fructose, in comparison with glucose, is only about 0.6%, i.e., even in the case of a large excess of fructose, the fructose is substantially not phosphorylated. Furthermore, mannose, galactose, glucosamine and N-acetyl-glucosamine do not react according to Equations 3 and 4 as given hereinafter. Since, in addition, in its preparation the new enzyme is obtained substantially free from phosphohexose isomerase and, in comparison with hexokinase, can be freed in a substantially much more simple manner from this and other disturbing enzymes (for example, glutathione-reductase, TPNH-oxidase, glucose-dehydrogenase), it is much more suitable for the enzymatic analysis of glucose I than is hexokinase.

As phosphate donor for the new enzyme, there can, according to the invention, be used not only the acetyl phosphate mentioned by Kamel et al. (supra) but also all phosphates having the following formula:

wherein R is an organic radical, which does not possess a negative charge in the immediate vicinity of the phosphate group. The new enzyme is, therefore, best designated acyl phosphate :glucose-6-phosphotransferase.

In the following Table 1, there are set out the relative velocities of the reaction of glucose with various phosphate donors yielding glucose-6-phosphate, in which the reaction is catalyzed by acyl phosphate: glucose-6-phosphotransferase. In the data, acetyl phosphate has been used as the comparison and as noted has been assigned a velocity value of 100.

TABLE 1 Relative Phosphate donor (p=PO3Hz) reactl on acyl phosphates veloclty Benzoyl phosphate C 160 0 Nicotoyl phosphate C 110 0 Aoetyl phosphate CH3G\ 100 0 Propionyl phosphate CH3-CHz-C\ 100 O 0 II N-benzoyl-glycoyl phosphate -C-NHCHz-C\ 90 0 Carbamyl phosphate H2NO\ 85 0 Glutaroyl phosphate HO O C-CHz-C H2CH2-C 11 0 Suoeinyl phosphate HO O C C Hz-C Hz-() 5 O P 0 11; O i 3-phosphoglycene acid l-phosphate HzC-CI-I( OH) --C\ 1. 3

Phosphate donor (p=PO;Hz) sugar phosphates Fructose6-phosphate 0 4. 5

p-O GH2 T011201;

Fructose-1,6-diphosphate O 1. 7

p-O -CHz wCHz-O-p Ribose-5-phosphate 0 0. 7

p-OCH2 W Glycerol-l-phosphate HO CH2-CH OHCH: O-p 0. 3

Glyceraldehyde-B-phosphate OHO-OHOH-C Hz-O-p 0 Glucose-l-phosphete HO-CHz 0 Glyceric acid-2-phosphate HOOC-(JH-CH OH 0 glyceric aeid-3-phosphate HO O CCH0HCH2O-p U Phosphe-enol-pyruvate H O O 0-? CH TABLE III.Contin ued Relative Phosphate donor (p=PO H reaction nucleoside phosphate velocity Adenosine-5-monophosphate 11111 N F K, N/

\/ CHz-O-D Adenosine-'-diphosphate IIIH: 0

N j N N w TCHz-O-p-p Adenosine-5-triphosphate- 1YIH2 0 N F l k A 2 -p-n-n Phosphate donor (p=PO3H2) other phosphoric acid derivatives Creatine phosphate /NH-p 0 EN C III-CH2C O OH 0113 Sodium pyrophosphate Na-p-p 0 Riboflavin-5'-acetyl phosphate Hg(J(CHOH);C Hz-O-p-O-fi-CH; 0

N N 0 H3O /\l/ k L NH From Table 1, it can be seen that the enzyme, i.e., acyl phosphate:glucose transferase preferentially reacts with those phosphate donors in which the eifect of a negative charge which may be present on the bond phosphate donor-enzyme is the smallest; see, for example, glutaroyl phosphate, succinyl phosphate and 3 phosphoglyceric acid-l-phosphate in comparison with benzoyl phosphate. When benzoyl phosphate is employed as phosphate donor, the enzyme reacts more rapidly than it does with acetyl phosphate.

This high afiinity of acyl phosphate:glucose-6-phosphotransferase for phosphate donors of general Formula I was surprising because the known phospho-transferases, such as the conventional hexokinase and phosphoglycerate-kinase will only react with nucleoside-triphosphates. Furthermore, it was not to have been foreseen that the new enzyme could be freed so easily from the accompanying enzymes, such as phosphohexose isomerase, glutathione-reductase, TPNH-oxidase, glucose dehydrogenase and the like, which have a disturbing action in glucose determinations and that it would, therefore, be

in which R is an organic radical, which radical does not possess a negative charge in the immediate vicinity of the phosphate group, and, as transferase, there is used acyl phosphate glucose-6-phosphotransferase.

The course of the reaction in the process according acyl phosphate: glucoseG-phosphotransferese (triphnsphopyridinenucleotide) (fi-phosphogluconic acid) 6PG TPNH H+ The new agents according to the present invention for use in the determination of glucose comprise a phosphate donor, a transferase, glucose-6-phosphate-dehydrogenase and triphosphopyridine-nucleotide and, advantageously, a buffer. The phosphate donor forming a component of the new agent is a phosphate having the Formula I and the transferase is acyl phosphate:glucose-6-phosphotransferase.

The process according to the present invention is carried out as follows:

The sample to be investigated is mixed with an acyl phosphate having the Formula I, triphosphopyridinenucleotide (TPN), a buffer (as for example, glycocoll buffer, triethanolamin buffer and tris-buffer) and glucose- 6-phosphate-dehydrogenase. Any glucose 6 phosphate which may be present will now react and can be determined beforehand. The acyl phosphate:glucose-6-phosphotransferase is then added and, after some minutes, the glucose is determined photometrically on the basis of the reduced triphosphopyridine nucleotide (TPNH) formed in the ensuing reaction. As this is an enzymatic reaction, the conventional precautions with respect to pH, temperature, etc., must all be observed. The preferred range of pH is between 8 and 9.5 and the preferred temperature is between 20 and 30 C. and should not exceed 35 C.

Since aqueous solutions of acyl phosphates of Formula I, which are, after all, mixed acid anhydrides, can only be stored for a limited period of time, the acyl phosphate required for use in a routine procedure can advantageously be synthesized immediately before theglucose determination in a preceding enzymatic reaction, preferably in the cuvette being used for the photometric determination.

Thus, for example, acetyl phosphate can be prepared from a stable acetate and ATP utilizing acetate-kinase and carbamyl phosphate can be prepared from carbamic acid and ATP using therefor carbamate-phosphokinase.

An especial advantage of the process according to the present invention in the enzymatic determination of glucose, in comparison with the previously known processes, is the high substrate specificity of the combination of acyl phosphate: glucose 6 phosphotransferase with glucose-6-phosphate-dehydrogenase. As can be seen from Table 2, which follows, the Michaelis constants (K which represents a measure of the afiinity for the substrate, for acyl phosphate: glucose 6-phosphotransferase differ, with regard to glucose and fructose, by a factor of 10 TABLE 2.MICHEALIS CONSTANTS FOR GLUCOSE AND FRUCTOSE WITH VARIOUS PHOSPHATE DONORS Acyl phosphate (Km) glucose (Km) fructose Acetyl phosphate mM.). 4. 10- M 5. ill- M Carhamyl phosphate (47 mM.) 5. 1O- M 3. IO- M Benzoyl phosphate (12 mM.) 8. 10 M 2. Ill- M Nicotoyl phosphate mM.) 5. 10- M 2. 10 M 8 EXAMPLE 1 Glucose-determination with carbamyl phosphate Fruit juice (black-currant) was analyzed for glucose as follows:

Reagents: End concentration 2.50 ml. of glycocoll bulfer having a pH of 9.0 (200 mM.) mM 0.10 ml. TPN (11 mM.; 10 mg./ml.) mM 0.37 0.10 ml. carbamyl phosphate (140 mM.; 20

mg./ml.) mM 4.7

(0.01 ml. of a 1:10 diluted sample and 2.97

ml. with water) 0.01 ml. glucose-6-phosphate-dehydrogenase (1 mg./ml.; 140 U/mg.) U/ml 0.47

Any G-6-P present reacted, and there was then added:

0.02 ml. acyl phosphate:glucose-6-phosphotransferase (1 mg./ml.:40 U/mg.) U/ml. 0.26

3.3 cm. /p. mol

366 mp d=layerthickness of the cuvette (1 cm.) V=test volume (3 ml.) v volume of the sample used In the determination of glucose, measured at 366 m there thus applied:

igluuose izluuuse 3.3.10 1?) 2) sample.

Thus, for the above example, the following result was obtained:

=mg. glucose/ml. of

.10=13.5 mg. glucose/ml. fruit juice EXAMPLE 2 Glucose determination with benzoyl phosphate The procedure was carried out in a manner analogous to that described in Example 1 but, in place of the carbamyl phosphate, there was used benzoyl phosphate (end molarity 3.3 mM.). The final value, following addition of acyl phosphate-transferase, was, as in Example 1, reached after 5 minutes. The same extinction value was observed.

EXAMPLE 3 Glucose determination with nicotoyl phosphate The measurement procedure was carried out as in Example 1 but using nicotoyl phosphate (end molarity 3.3 mM.) in place of the carbamyl phosphate. In this case, a 0.01 M a-D-glucose solution was prepared and analyzed. In the glucose determination, there were used 0.02

7 ml. of this latter solution of (=0.2,u mol=0.036 mg.

glucose). Following introduction of the acyl phosphate: glucose-6-phosphotransferase, the end value was determined after 5 minutes with AE=0.222.

=mg. glucose/n11. 0.222.0.164 0.02 which corresponded to 0.0364 mg. glucose/ml. tested, i.e., the determination corresponded to 101%.

= 1.82 mg. glucose/ml. of the solution used 9 EXAMPLE 4 Glucose determination with acetyl phosphate formation in the cuvette The reaction was started with:

(0.02 ml. of a 1:10 diluted dietetic fruit juice (black-currant) and 2.96 ml. with water) The end value for glucose was reached after 8 minutes. AE at 366 m t=0.l70. Calculation according to Example 1 gave:

O -10=13.9 mg. glucose/ml. fruit iuico EXAMPLE Glucose determination in blood with acetyl phosphate Ten 0.1 ml. samples of different blood specimens were each mixed with 1 ml. 0.33 M perchloric acid in order to deproteinize the same. After centrifuging, 0.2 ml. samples were added to the test mixture. The further procedure followed was that set out in Example 1, other than that acetyl phosphate (end molarity 4 mM.) was used as phosphate donor instead of carbamyl phosphate. By calculation as in Example 1 there were obtained the values set out in Table 3 which follows which table for the purposes of comparison also gives the glucose values of the same test samples determined by the hexokinaseintermediate ferment method.

acyl phosphate: Mg percent glucose-6ph0sphowith HK/ Deviation, transferase G G-PDH percent EXAMPLE 6 Glucose and fructose determinations in blood to which increasing amounts of fructose were added Before de-proteinization, two different blood samples were mixed with different amounts of fructose (set out in Table 4). The further procedure was that described in Example 5. In addition, fructose was determined by the method of H. Klotzsch and H. U. Bergmeyer (cf. H. U. Bergmcycr, Methoden der enzymatischen Analyse,

10 Verlag Chemie, Weinheim, 1962, page 156). The results obtained are set out in Table 4 which follows:

TABLE 4.GLUCOSE AND FRUCTOSE DETERMINATIONS IN BLOOD IN WHICH INCREASING AMOUNTS OF FRUC- TOSE WERE ADDED TO THE BLOOD Glucose, mg. percent With acyl phosphate:

glucose-6- phospho- With Difference Fructose transferase HK in percent;

Number EXAMPLE 7 Glucose determinations in test materials having a high fructose content Measurements were carried out in the manner described in Example 1 but using in place of carbamyl phosphate 4 mM acetyl phosphate as the phosphate donor. The smooth manner in which the measurement could be carried out is particularly noticeable. Even traces of glucose in very pure fructose could be detected with certainty by the method according to the present invention. The results obtained are set out in Table 5 which follows:

TABLE 5.GLUOOSE DETERMINATION IN TEST MATERI- ALS HAVING A HIGH FRUCTOSE CONTENT 1 About 200 mg. fructose/ml. were added to the juice, thereby slightly diluting it.

2 About 100.00. 3 In this case, the procedure could not be carried out.-

We claim: 1. Process for enzymatic determination of glucose in a sample containing glucose and fructose which comprises mixing said sample with a phosphate donor having the formula:

wherein R is an organic radical which does not possess a negative charge in the vicinity of the phosphate group and acyl phosphate:glucose-6-phosphotransferase, thereafter reacting the glucose-6-phosphate formed with triphosphopyridinenucleotide (TPN) and glucoSe-6-phosphate-dehydrogenase to form reduced triphosphopyridine nucleotide and spectrophotometrically determining the glucose present on the basis of the reduced triphosphopyridine-nucleotide.

2. Process according to claim 1, wherein said phosphate donor is a member selected from the group consisting of benzoyl phosphate, carbamyl phosphate, nicotoyl phosphate, acetyl phosphate, propionyl phosphate, and N- benzoyl-glycol phosphate.

3. Process according to claim 1, wherein said phosphate donor is nicotoyl phosphate.

4. Process according to claim 1, wherein said phosphate donor is acetyl phosphate.

5. Process according to claim 1, wherein said phos gahate donor is benzoyl phosphate.

'6. Process according to claim 1, wherein said phosphate donor is prepared just prior to said reaction by reacting adenosine-triphosphate, acylate ions, and an acylate phosphokinase.

7. An agent for use in the enzymatic determination of glucose in a sample containing glucose and fructose, which comprises a phosphor donor selected from the group consisting of nicotoyl phosphate and benzoyl phosphate; acyl phosphate:glucose-6-phosphotransferase; glucose-6-phosphate-dehydrogenase; and triphosphopyridine-nucleotide.

8. Agent according to claim 7 additionally containing a buffer.

9. Agent according to claim 7, wherein in place of said phosphate donor there is employed a mixture of adenosinetriphosphate; an acylate ion selected from the group consisting of benzoyl ions and nicotoyl ions; and the corresponding acylate-phosphokinase.

References Cited Karnel et al.: J. Biol. Chem., vol. 239, pp. PC3607 and PC3608 (1964).

Rose et al.: J. Biol. Chem, vol. 211, pp. 737, 738, 748, 749, 754, 755 (1954).

ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 509 O25 Dat d April 28 1970 Inventor) Hans Ulrich Bergmeyer et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1 formula following line 19 II I 0-C-OPO H should read R-C-(J-PO H Column 9 lines 23 and 24 "(0. 02 ml of a 1:10 diluted dietetic fruit juice (black current) and 2. 96 ml. with water) should read 0 02 ml acyl phosphate glucose -6- phosphotransferase (l mg./ml 40 U/mg.) 0 26 U/ml.

Signed and sealed this 29th day of December 1970 (SEAL) Attest:

EDWARD M.FLETCHER,JR. WILLIAM E. SCHUYLER, JR. Attesting Officer Commissioner of Patents FORM PO-- O5O (10-69) uscoMM-Dc 0037a pus Q U 5, GOVERNMENT HUNTING OFFICE: I91! OJB 33 

